Normal human diploid cells have a finite potential for proliferative growth (Hayflick, L. et al., Exp. Cell Res. 25:585 (1961); Hayflick, L., Exp. Cell Res. 37:614 (1965)). Indeed, under controlled conditions in vitro cultured human cells can maximally proliferate only to about 80 cumulative population doublings. The proliferative potential of such cells has been found to be a function of the number of cumulative population doublings which the cell has undergone (Hayflick, L. et al., Exp. Cell Res. 25:585 (1961); Hayflick, L. et al., Exp. Cell Res. 37:614 (1985)). This potential is also inversely proportional to the in vivo age of the cell donor (Martin, G. M. et al., Lab. Invest. 23:86 (1979); Goldstein, S. et al., Proc. Natl. Acad. Sci. U.S.A.) 64:155 (1969); Schneider, E. L., Proc. Natl. Acad. Sci. (U.S.A.) 73:3584 (1976); LeGuilty, Y. et al., Gereontologia 19:303 (1973)).
Cells that have exhausted their potential for proliferative growth are said to have undergone "senescence." Cellular senescence in vitro is exhibited by morphological changes and is accompanied by the failure of a cell to respond to exogenous growth factors. Cellular senescence, thus, represents a loss of the proliferative potential of the cell. Although a variety of theories have been proposed to explain the phenomenon of cellular senescence in vitro, experimental evidence suggests that the age-dependent loss of proliferative potential may be the function of a genetic program (Orgel, L. E., Proc. Natl. Acad. Sci. (U.S.A.) 49:517 (1963); De Mars, R. et al., Human Genet. 16:87 (1972); M. Buchwald, Mutat. Res. 44:401 (1977); Martin, G. M. et al., Amer. J. Pathol. 74:137 (1974); Smith, J. R. et al., Mech. Age. Dev. 13:387 (1980); Kirkwood, T. B. L. et al., Theor. Biol. 53:481 (1975).
Cell fusion studies with human fibroblasts in vitro have demonstrated that the quiescent phenotype of cellular senescence is dominant over the proliferative phenotype (Pereira-Smith, O. M. et al., Somat. Cell Genet. 8:731 (1982); Norwood, T. H. et al., Proc. Natl Acad. Sci. (U.S.A.) 71:223 (1974); Stein, G. H. et al., Exp. Cell Res. 130:155 (1979)).
Insight into the phenomenon of senescence has been gained from studies in which senescent and young (i.e. non-senescent) cells have been fused to form heterodikaryons. In order to induce senescence in the "young" nucleus of the heterodikaryon (as determined by an inhibition in the synthesis of DNA), protein synthesis must occur in the senescent cell prior to fusion (Burmer, G. C. et al., J. Cell. Biol. 94:187 (1982); Drescher-Lincoln, C. K. et al., Exp. Cell Res. 144:455 (1983); Burner, G. C. et al., Exp. Cell. Res. 145:708 (1983); Drescher-Lincoln, C. K. et al., Exp. Cell Res. 153:208 (1984).
Likewise, microinjection of senescent fibroblast mRNA into young fibroblasts has been found to inhibit both the ability of the young cells to synthesize DNA (Lumpkin, C. K. et al., Science 232:393 (1986)) and the ability of the cells to enter into the S (stationary) phase of the cell cycle (Lumpkin, C. K. et al., Exp. Cell Res. 160:544 (1985)). Researchers have identified unique mRNAs that are amplified in senescent cells in viro (West, M. D. et al., Exp. Cell Res. 184:138 (1989); Giordano, T. et al., Exp. Cell Res. 185:399 (1989)).
The human diploid endothelial cell presents an alternative cell type for the study of cellular senescence because such cells mimic cellular senescence in vitro (Maciag, T. et al., J. Cell. Biol. 91:420 (1981); Gordon, P. B. et al., In Vitro 19:661 (1983); Johnson, A. et al., Mech Age. Dev. 18:1 (1982); Thornton, S. C. et al., Science 222:623 (1983); Van Hinsbergh, V. W. M. et al., Eur. J. Cell Biol. 42:101 (1986); Nichols, W. W. et al., J. Cell. Physiol. 132:453 (1987)).
In addition, the human endothelial cell is capable of expressing a variety of functional and reversible phenotypes. The endothelial cell exhibits several quiescent and non-terminal differentiation phenotypes (Folkman, J. et al., Nature 288:551 (1980); Maciag, T. et al., J. Cell Biol. 94:511 (1982); Madri. J. A. et al., J. Cell Biol. 97:153 (1983); Montesano, R., J. Cell Biol. 99:1706 (1984); Montesano, R. et al., J. Cell Physiol. 34:460 (1988)).
It has been suggested that the pathway of human cell differentiation in vitro involves the induction of cellular quiescence mediated by cytokines that inhibit growth factor-induced endothelial cell proliferation in vitro (Jay, M. et al., Science 228:882 (1985); Madri, J. A. et al., In Vitro 23:387 (1987); Kubota, Y. et al., J. Cell Biol. 107:1589 (1988); Ingber, D. E. et al., J. Cell Biol. 107:317 (1989)).
Inhibitors of endothelial cell proliferation also function as regulators of immediate-early transcriptional events induced during the endothelial cell differentiation in vitro, which involves formation of the capillary-like, tubular endothelial cell phenotype (Maciag, T., In: Imp. Adv. Oncol. (De Vita, V. T. et al., eds. , J.B. Lippincott. Philadelphia, 42 (1990); Goldgaber, D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7606 (1990); Hla, T. et al., Biochem Biophys. Res. Commun. 167:637 (1990)). The inhibitors of cell proliferation that include: 1. Interleukin-la (IL-1a) (Montesano, R. et al., J. Cell Biol. 99:1706 (1984); Montesano, R. et al., J. Cell Physiol. 122:424 (1985); Maciag, T. et al. (Science 249:1570-1574 (1990));
2. Tumor necrosis factor (Frater-Schroder, M. et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5277 (1987); Sato, N. et al., J. Natl. Cancer Inst. 76:1113 (1986); Pber, J. P., Amer. J. Pathol. 133:426 (1988); Shimada, Y. et al., J. Cell Physiol. 142:31 (1990));
3. Transforming growth factor-.beta. (Baird, A. et al., Biochem. Biophys. Res. Commun. 138:476 (1986); Mullew, G. et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5600 (1987); Mairi, J. A. et al., J. Cell Biol. 106:1375 (1988));
4. Gamma-interferon (Friesel, R. et al., J. Cell Biol. 104:689 (1987); Tsuruoka, N. et al., Biochem. Biophys. Res. Commun. 155:429 (1988)) and
5. The tumor promoter, phorbol myristic acid (PMA) (Montesano, R. et al., Cell 42:469 (1985); Doctrow, S. R. et al., J. Cell Biol. 104:679 (1987); Montesano, R. et al, J. Cell. Physiol. 130:284 (1987); Hoshi, H. et al., FASAB J. 2:2797 (1988)).
The prospect of reversing senescence and restoring the proliferative potential of cells has implications in many fields of endeavor. Many of the diseases of old age are associated with the loss of this potential. Also the tragic disease, progeria, which is characterized by accelerated aging is associated with the loss of proliferative potential of cells. Restoration of this ability would have far-reaching implications for the treatment of this disease, of other age-related disorders, and, of aging per se.
In addition, the restoration of proliferative potential of cultured cells has uses in medicine and in the pharmaceutical industry. The ability to immortalize nontransformed cells can be used to generate an endless supply of certain tissues and also of cellular products.
The significance of cellular senescence has accordingly been appreciated for several years (Smith, J. R., Cellular Ageing, In: Monographs in Development Biology; Sauer, H. W. (Ed.), S. Karger, New York, N.Y. 17:193-208 (1984); Smith, J. R. et al., Exper. Gerontol. 24:377-381 (1989), herein incorporated by reference). Researchers have attempted to clone genes relevant to cellular senescence. A correlation between the existence of an inhibitor of DNA synthesis and the phenomenon of cellular senescence has been recognized (Spiering, A.I. et al., Exper. Cell Res. 179:159-167 (1988); Pereira-Smith, O. M. et al., Exper. Cell Res. 160:297-306 (1985); Drescher-Lincoln, C. K. et al., Exper. Cell Res. 153:208-217 (1984); Drescher-Lincoln, C. K. et al., Exper. Cell Res. 144:455-462 (1983)). Moreover, the relative abundance of certain senescence-associated RNA molecules has been identified (Lumpkin, C. K. et al., Science 232:393-395 (1986)).
Several laboratories have used the "subtraction-differential" screening method to identify cDNA molecules derived from RNA species that are preferentially present in senescent cells (Kleinsek, D. A., Age 12:55-60 (1989); Giordano, T. et al., Exper. Cell. Res. 185:399-406 (1989); Sierra, F. et al., Molec. Cell. Biol. 9:5610-5616 (1989); Pereira-Smith, O. M. et al., J Cell. Biochem. (Suppl 0 (12 part A)) 193 (1988); Kleinsek, D. A., Smith, J. R., Age 10:125 (1987)).
In one method, termed "subtraction-differential" screening, a pool of cDNA molecules is created from senescent cells, and then hybridized to cDNA or RNA of growing cells in order to "subtract out" those cDNA molecules that are complementary to nucleic acid molecules present in growing cells. Although useful, for certain purposes, the "subtraction-differential" method suffers from the fact that it is not possible to determine whether a senescence-associated cDNA molecule is associated with the cause of senescence, or is produced as a result of senescence. Indeed, many of the sequences identified in this manner have been found to encode proteins of the extra-cellular matrix. Changes in the expression of such proteins would be unlikely to cause senescence.